D-alanine racemase mutants of mycobacteria and uses therefore

ABSTRACT

The present invention is directed to D-alanine racemase mutants of mycobacterial species. The D-alanine racemase gene (alrA) is involved in the systhesis of D-alanine, a basic component of peptidoglycan that forms the backbone of the bacterial cell wall. The present invention is also directed to methods of making live-attenuated vaccines against pathogenic mycobacteria using such alrA mutants and to the vaccines made according to such methods. The present invention is further directed to use of alrA mutants in methods for screening antimycobacterial agents that are synergistic with peptidoglycan inhibitors. Finally, the present invention is directed to methods to identify new pathways of D-alanine biosynthesis for use in developing new drugs targeting peptidoglycan biosynthesis in mycobacteria and to identify vaccines useful against pathogenic mycobacteria.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is related to provisional patentapplication Serial No. 60/341,485, filed Dec. 18, 2001, which isincorporated herein by reference.

STATEMENT REGARDING GOVERNMENT RIGHTS

[0002] This invention was made with government support from UnitedStates Department of Agriculture, USDA Cooperative State ResearchService Project Grant No. NEB. 14-108. The Government may have certainrights in this invention.

BACKGROUND OF THE INVENTION

[0003] The present invention is directed to D-alanine racemase mutantsof mycobacterial species. The D-alanine racemase gene (alrA) is involvedin the synthesis of D-alanine, a basic component of peptidoglycan thatforms the backbone of the bacterial cell wall. The present invention isalso directed to methods of making live-attenuated vaccines againstpathogenic mycobacteria using such alrA mutants and to the vaccines madeaccording to such methods. The present invention is further directed touse of alrA mutants in methods for screening antimycobacterial agentsthat are synergistic with peptidoglycan inhibitors. Finally, the presentinvention is directed to methods to identify new pathways of D-alaninebiosynthesis for use in developing new drugs targeting peptidoglycanbiosynthesis in mycobacteria and to identify vaccines useful againstpathogenic mycobacteria.

[0004] The publications and other materials used herein to illuminatethe background of the invention or provide additional details respectingthe practice, are incorporated by reference, and for convenience arereferenced in the following text by author and date and are listedalphabetically by author in the appended bibliography.

[0005] Mycobacteria cause a number of diseases in humans and animalsincluding tuberculosis, which is the leading cause of human death froman infectious disease in the world (Bloom & Murray, 1992). M.tuberculosis is the principal cause of tuberculosis in humans and otherprimates and is occasionally seen in dogs, pigs and cattle (O'Reilly &Daborn, 1995). In contrast, M. bovis, the etiologic agent of bovinetuberculosis, has a wide host range and infects ruminants, carnivores,and primates, including humans (Pritchard, 1988). For M. bovis and M.tuberculosis in both humans and animals, contaminated aerosols are themost common routes of transmission (Carleton, 1993). Other mycobacterialpathogens of importance are M. avium, M. paratuberculosis, and M.leprae. M. avium is the agent of tuberculosis in birds but its majorsignificance is as an opportunistic pathogen of AIDS patients (Inderliedet al., 1993). M. paratuberculosis is the etiologic agent of Johne'sdisease, a granulomatous enteritis in ruminants and it has also beenlinked to a potential etiology of a type of inflammatory bowel disease(Crohn's disease) in humans (Cocito et al., 1994; Harris and Barletta,2001). Finally, M. leprae infects humans and armadillos. Though leprosyin humans has low mortality, its morbidity is quite high in affectedareas, estimated to have been 10-12 million in the 1980s (Noordeen,1991). All these diseases caused by mycobacteria are characterizedpathologically by the formation of granulomatous nodules (tumor-likemasses caused by chronic inflammatory processes) or “tubercules” thatare seen in advanced cases. Due to the significance of mycobacterialdiseases, prevention and control measures, including vaccines,diagnostics and therapies are of major importance.

[0006] Microorganisms of the M. avium complex have achieved prominenceas major opportunistic pathogens of AIDS patients. M avium is naturallyresistant to most firstline antituberculosis drugs (Inderlied et al.,1993). This threat to public health has been partially met by therapywith appropriate antimicrobial agents, but unfortunately drug-resistantM. avium and M. tuberculosis strains readily appear (Chaisson et al.,1994; Espinal et al., 2000), underscoring the need to develop new andmore effective anti-mycobacterial agents.

[0007]Mycobacterium smegmatis is a fast-growing nonpathogenicmycobacterial species particularly useful in studying basic cellularprocesses of relevance to pathogenic mycobacteria. The D-alanineracemase gene (alrA) is involved in the synthesis of D-alanine, a basiccomponent of peptidoglycan that forms the backbone of the cell wall.Biosynthesis of the mycobacterial cell wall has received considerableattention in the search for inhibitors useful for drug therapy(Chatterjee, 1997). These cell walls display a complex architecture ofglycolipids and proteins linked to themycolyl-arabinogalactan-peptidoglycan backbone (McNeil and Brennan,1991). This structure is a barrier that contributes to drug resistance(Trias and Benz, 1994), and many of its components have been found toplay a major role in pathogenesis (Daffe and Draper, 1998). The analysisof the M. tuberculosis genome sequence suggests that peptidoglycanbiosynthesis in mycobacteria follows the general pathway of otherbacteria, including the formation of the basic building blockD-alanyl-D-alanine (Belanger and Inamine, 2000; Cole et al., 1998).D-alanine racemase (Air) catalyzes the conversion of L-alanine intoD-alanine (Julius et al., 1970), and D-alanine-D-alanine ligasecatalyzes the subsequent dimerization of D-alanine into the keydipeptide D-alanyl-D-alanine (Neuhaus, 1962). The corresponding enzymesfrom both Escherichia coli (Lambert and Neuhaus, 1972; Neuhaus andLynch, 1964) and mycobacteria (Cáceres, 1999; David et al., 1969) areinhibited by D-cycloserine (DCS), a D-alanine analog (Neuhaus, 1967).The dipeptide is then added to the UDP-tripeptide precursor by theaction of the D-alanine-D-alanine adding enzyme that completes thereactions of the D-alanine branch of peptidoglycan assembly (Walsh,1989).

[0008] DCS is particularly effective against mycobacteria albeit withmarked side effects (Cummings et al., 1955; Yew et al., 1993). Moreover,overproduction of Air in Mycobacterium smegmatis, Mycobacteriumintracellulare, and Mycobacterium bovis BCG leads to a DCS-resistantphenotype. The M. smegmatis enzyme is inhibited by DCS in aconcentration-dependent manner (Cáceres et al., 1997). Likewise, the M.avium and M tuberculosis enzymes produced from E. coli recombinantclones are also inhibited by DCS (Strych et al., 2001). Nonetheless, thespecific characteristics of the mycobacterial enzymes involved inpeptidoglycan biosynthesis, including the essentiality of each of theirfunctions, remain unknown. Such knowledge would be important to thedesign of specific inhibitors that would serve as novel bactericidalagents to treat M. tuberculosis and M avium infections. Furthermore, theinactivation of the genes encoding for these enzymes may lead to thegeneration of attenuated strains of pathogenic mycobacteria that couldserve as candidate vaccines against tuberculosis.

[0009]M. smegmatis has been extensively used as a model system for Mtuberculosis and other pathogenic mycobacteria. M smegmatis isnonpathogenic, requiring less stringent containment facilities, and itgrows at a relatively high rate in a variety of defined andnutrient-restricted media (Jacobs, 2000). M smegmatis has been used tostudy drug resistance mechanisms (Cáceres et al., 1997; Peteroy et al.,2000; Telenti et al., 1997) and basic physiological processes includingthe synthesis of peptidoglycan precursors (Cirillo et al., 1998; Pavelkaand Jacobs, 1996). Insights gained from these studies can then beapplied to the pathogenic mycobacteria.

SUMMARY OF THE INVENTION

[0010] It is an object of the present invention to provide a D-alanineracemase mutant mycobacteria which can be used in development oftreatments for diseases caused by pathogenic mycobacteria.

[0011] Accordingly, in one aspect of the present invention, a novelmycobacterial strain, comprising a D-alanine racemase mutant which isnot dependent on D-alanine for growth, designated alrA mutant, isprovided. The mycobacterial strain is generated from a pathogenicmycobacterium. Any pathogenic mycobacteria may be used in accordancewith the present invention. Preferred pathogenic mycobacteria are M.tuberculosis, M. bovis, and all subspecies of M. avium.

[0012] In another aspect of the invention, a live-attenuated vaccineagainst pathogenic mycobacteria is provided which includes the alrAmutant, with or without additional mutations. In one embodiment of thisaspect, the alrA mutant displays increased susceptibility to anantimycobacterial agent. In a second embodiment, the alrA mutantdisplays increased susceptibility to bacterial action of phagocyticcells. In a third embodiment, the mutant is a D-alanine dependentpathogenic mycobacterium.

[0013] In a further aspect of the present invention, methods forproducing live-attenuated vaccines including the alrA mutant areprovided.

[0014] In another aspect of the invention, a method for screeningantimycobacterial agents that are synergistic with peptidoglycaninhibitors is provided.

[0015] In yet a further aspect of the invention, a method to identifynew pathways of D-alanine biosynthesis is provided. The pathways soidentified may be used to develop new drugs that target peptidoglycanbiosynthesis in mycobacteria and to identify new vaccines useful againstpathogenic mycobacteria.

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention is directed to D-alanine racemase mutantsof mycobacterial species. The D-alanine racemase gene (alrA) is involvedin the synthesis of D-alanine, a basic component of peptidoglycan thatforms the backbone of the bacterial cell wall. The present invention isalso directed to methods of making live-attenuated vaccines againstpathogenic mycobacteria using such alrA mutants and to the vaccines madeaccording to such methods. The present invention is further directed touse of alrA mutants in methods for screening antimycobacterial agentsthat are synergistic with peptidoglycan inhibitors. Finally, the presentinvention is directed to methods to identify new pathways of D-alaninebiosynthesis for use in developing new drugs targeting peptidoglycanbiosynthesis in mycobacteria and to identify vaccines useful againstpathogenic mycobacteria.

[0017] The present invention employs the following definitions.

[0018] “alrA” and “alr” refer to the D-alanine racemase gene, includingnormal alleles of the alrA gene.

[0019] “AlrA” and “Alr” refer to D-alanine racemase enzyme.

[0020] “Displays increased susceptibility to antimycobacterial agent”refers to a reduction in the minimal inhibitory concentration of themutant strain when compared with the wild type strain.

[0021] “Displays increased susceptibility to bactericidal action ofphagocytic cells” refers to a reduction in the ability of the mutantstrain to survive and/or replicate in phagocytic cells when compared tothe wild type strain.

[0022] A “promoter” is a DNA sequence that directs the transcription ofa structural gene. Typically, a promoter is located in the 5′ region ofa gene, proximal to the transcriptional start site of a structural gene.If a promoter is an inducible promoter, then the rate of transcriptionincreases in response to an inducing agent. For example, a promoter maybe regulated in a tissue-specific manner such that it is only active intranscribing the associated coding region in a specific tissue type(s).

[0023] In contrast, the rate of transcription is not generally regulatedby an inducing agent if the promoter is a constitutive promoter. Thepromoter may be tissue-geneal, also known as non-tissue-preferred, suchthat it is active in transcribing the associated coding region in avariety of different tissue types.

[0024] It has been discovered that the M. smegmatis alrA gene can beinsertionally inactivated to generate AlrA null mutants. Unexpectedly,the inactivation of the alrA gene in M. smegmatis mutant did not resultin a growth requirement for D-alanine. Given the conservation of basicprocesses within the genus Mycobacterium, a similar outcome cane beexpected in all mycobacterial species. PCR and Southern blottinganalyses of wild-type and mutant strains confirmed the inactivation ofthe alrA gene in TAM2O and TAM23 by gene replacement via adouble-crossover event between the M. smegmatis chromosome and thelinear fragment carrying the inactivated gene. Thus, alrA mutants areviable and can grow in medium without D-alanine. Complementation of thealrA mutant strain with a wild-type alrA copy restores the wild-typephenotype, indicating that properties of the mutant strain are due tothe inactivation of the alrA gene rather than a polar effect on theexpression of a downstream gene. The independence of M. smegmatis alrAmutants of D-alanine for growth indicates that this mutation does notimpose an auxotrophic requirement for D-alanine. This phenotype hassignificant implications for the synthesis of D-alanine andpeptidoglycan in M smegmatis and for other mycobacterial species.

[0025] D-Alanine is an essential component for bacteria with apeptidoglycan layer structure. The essentiality of D-alanine stems fromthe key role of the dipeptide D-alanyl-D-alanine in the cross-linking ofpeptidoglycan strands (Strominger, 1962). The repeating unit of thepeptidoglycan from M smegmatis has a D-alanine moiety (Petit et al.,1969), and D-alanyl-D-alanine is the only product detected in abiochemical assay using partially purified extracts of M smegmatisD-alanine ligase (Peteroy et al., 2000). These properties support thehypothesis that D-alanine is also an essential component for Msmegmatis. In the context of this hypothesis, the independence of alrAmutants of D-alanine for growth suggests that M. smegmatis may havealternative pathways for the biosynthesis of D-alanine. Listeriamonocytogenes, for example, has been shown to possess such metabolicroutes (Thompson et al., 1998).

[0026] As described in further detail herein, alrA mutant mycobacteriacan be used to develop safe live-attenuated vaccines of pathogenicmycobacteria, screen for drugs that act synergistically withpeptidoglycan inhibitors, and identify new pathways of D-alaninebiosynthesis. In summary, the D-alanine independent phenotype of Msmegmatis alrA mutants suggests that M smegmatis has another pathway ofD-alanine biosynthesis. Given the conservation of basic physiologicalprocesses, this finding is of significance for pathogenic mycobacteriaand the design of novel attenuated strains and antimycobacterial agents.

[0027] Method of Use: Development of Novel alrA Mutant MycobacterialStrains

[0028] Novel alrA mutant pathogenic mycobacterial strains can begenerated in accordance with the present invention. The generation ofalrA mutants has been illustrated by preparation of M. smegmatis alrAmutants. However, other methods known in the art may be used to generatestable alrA mutants such as chemical mutagenesis followed by mutantenrichment, transposon mutagenesis, plasmid or phage vectors, andmethods to produce unmarked in-frame deletions. The same proceduresdescribed herein may be used to inactivate the alrA gene of otherpathogenic mycobacterial species in order to generate alrA mutants ofsuch species. Such species of mycobacteria include Mycobacteriumtuberculosis, Mycobacterium avium, Mycobacterium bovis, Mycobacteriumafricanum, and Mycobacterium paratuberculosis To inactivate the alrAgene in M smegmatis, a DNA fragment carrying the wild-type gene wassubcloned into the E. coli cloning vector pBlue-script II KS(+) to yieldthe recombinant plasmid pTAMU1 (Table 1). Then, the 1.2-kb kanamycinresistance determinant from plasmid pUC4K containing the aminoglycoside3′-phos-photransferase type I-coding gene from transposon Tn9O3 (30) wasinserted at the unique PstI site internal to the alrA gene. Theresulting construct, pTAMU2, carries an insertionally in-activated alrAgene, which can be excised as a linear 3.2-kb BamHI-kvnl fragment (Table1). Based on the hypotheses that D-alanine is an essential component andthat AlrA is the only enzyme responsible for D-alanine biosynthesis in Msmegmatis, it was predicted that an alrA mutant would be dependent onD-alanine for growth. Unexpectedly, the inactivation of the alrA gene inM. smegmatis mutant did not result in a growth requirement forD-alanine. The step to select for this mutant was carried out in MADCagar supplemented with 50 mM D-alanine in addition to 20 ug of kanamycinper ml. Transformation of M smegmatis mc²155 with the 3.2-kb linearfragment carrying the inactivated alrA gene yielded 25kanamycin-resis-tant transformants, and 2 of these, designated TAM20 andTAM23, were further analyzed. TABLE 1 Strains and plasmids Strain orSource or plasmid Relevant characteristics reference E. coli DH5aF⁻lacZDM15 endA1 hsdR17 supE44 Invitrogen gyrA96 relA1 Life Tech-nologies E. coli D(mcrA)183 D(mcrCB-hsdSMR-mrr)173 Stratagene XL2-BlueMRF′ endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F′ proAB lacI^(q)ZDM15Tn10 Tet^(R) Cam^(R)] M. smegmatis Alr⁺, high efficiency plasmid (37)mc²155 transformation mutant of M. smegmatis mc²6 M. smegmatisAlr⁻Kan^(R), M. smegmatis alr mutant This study TAM20 derived frommc²155 M. smegmatis Alr⁻Kan^(R), M. smegmatis alr mutant This studyTAM23 derived from mc²155 M. smegmatis Alr⁺Hyg^(R)Kan^(R), M. smegmatisalr mutant This study TAM23 complemented with wild type gene (pTAMU3)integrated at the mycobacteriophage L5 attB site pBUN82 Kan^(R) ,recombinant plasmid carrying the (5) alr gene from M. smegmatis mc²155in a 1.9 kb BamHI/PvuII fragment pBluescript Amp^(R), standard E. colicloning vector Stratagene II KS + pTAMU1 Amp^(R), pBluescript II KS +with This study the 1.9 kb BamHI/PvuII fragment of pBUN82 in theBamHI/EcoRV site pTAMU2 Amp^(R) Kan^(R), pTAMU1 with the This study 1.24kb PstI aph fragment of pUC4K (Pharmacia, Inc. Piscataway, N. J.) in thePstI site pTAMU3 Amp^(R)Kan^(R), pYUB412 with the This study 1.9 kbBamHI/PvuII fragment of pBUN82 in the BclI/EcoRV site pYUB412Amp^(R)Hyg^(R), E. coli-Mycobacterium (33) integration-proficientvector. Integrates at the mycobacteriophage L5 attB site

[0029] To determine whether these transformants carry an inactivatedalrA gene, genomic DNA was isolated and amplified by PCR. As expectedfor the inactivation of the alrA gene, genomic DNA from both TAM2O andTAM23 yielded the 2.4-kb product. Southern blotting analysis was used toverify the occurrence of these recombinational events in the appropriateM. smegmatis strains. Genomic DNA was isolated; digested with SmaI,which cuts once within the alrA gene; transferred to a membrane; andhybridized with the wild-type alrA gene fragment as a probe. Thewild-type strain mc²155 gave two homologous bands of approximately 15.0and 1.8 kb, whereas TAM20 and TAM23 yielded a mutant-type pattern withthree bands of approximately 15.0, 2.2, and 1.2 kb. These patterns wereas expected for the predicted recombinational events, validating theconstruction of the strains described herein.

[0030] The identification of only one D-alanine racemase gene in themycobacterial genome sequencing projects suggests that M. smegmatis alrAmutants may be dependent on exogenous D-alanine for growth. Both mutantstrains, TAM2O and TAM23, exhibited wild-type growth in MADC agarsupplemented with D-alanine, giving rise to typical flat-border coloniesafter 3 days of incubation at 37C. In the absence of D-alanine, TAM2Oand TAM23 cells were also able to grow, but colonies displayed a drierappearance with more raised borders. Complementation of TAM23 with theintegrating construct pTAMU3 introduces a wild-type alrA gene at themycobacteriophage L5 attachment site (Lee and Hatfull, 1993; Pascopella,1994) and fully restores wild-type colony morphology. Except for thesedifferences in colony morphology, no other observable differences weredetected by light or electron microscopy when cells were grown in thepresence or absence of D-alanine. Bacilli from both wild-type and mutantstrains were weakly gram positive, acid fast, and displayed the sameaspect of elongated rods. Likewise, at the ultrastructural level, cellsdid not differ in either shape, size, or thickness of the cell walls. Insummary, M smegmatis alrA mutants are independent of exogenousD-alanine, a property that was further confirmed by their ability togrow in MADC, as described more fully herein, and minimal brothcontaining mineral salts, glycerol, pyridoxal phosphate, and Tween 80.

[0031] To determine whether M smegmatis alrA mutants are or are notdevoid of Alr activity, crude cell extracts from wild-type and mutantstrains were prepared and assayed for enzyme activity. Protein extractsprepared from wild type mc²155 cells grown in the presence or absence ofD-alanine displayed approximately equal levels of Alr activity thatmatched the specific activities previously reported (Cáceres et al.,1997). In contrast, extracts from TAM23 prepared from cells grown in thepresence or absence of D-alanine lacked any detectable Alr activity.These extracts yielded background levels of Alr activity notsignificantly different from the levels obtained by replacing bovineserum albumin for TAM23 extracts in the reaction mixture. This absenceof Alr activity in TAM23 extracts was not due to the presence of aninhibitor since mixtures of TAM23 and mc²155 extracts gave the Alractivity proportional to the amount of enzyme present in the activeextract from the wild-type strain. Furthermore, to rule out thepossibility that sonic disruption may have damaged more readily proteinextracts of potentially fragile TAM23 cells, LDH activity was alsomeasured. The M smegmatis LDH activity was found to be more sensitive toprolonged sonic disruption and provides a useful control to determinewhether TAM23 extracts are enzymatically active. In contrast to theresults obtained in the Alr assay, extracts from both mc²155 and TAM23displayed similar levels of LDH activity above back-ground levels.Furthermore, complementation of TAM23 with a wild-type air gene restoredAlr activity. Thus, it was concluded that inactivation of the alrA generesults in no detectable Alr activity in the M. smegmatis alrA mutantstrain.

[0032] Method of Use: Generation of Live-Attenuated Vaccines

[0033] An ideal vaccine against diseases caused by mycobacteria such astuberculosis, leprosy, and Crohn's disease in humans and Johne's diseasein dairy cattle should be able to protect against the widest possiblerange of pathogenic bacterial isolates eliciting a strong CMI requiringpresentation of both peptide and non-peptide antigens, long-lastingimmunological memory, and possibly humoral immunity. Given thiscomplexity, live attenuated vaccines may offer the best possibilitiesfor an ideal antimycobacterial vaccine. Furthermore, this type ofvaccine could potentially be administered orally as enteric-coatedlyophilized tablets, thus avoiding the use of needles (Barletta et al.,1990). Additionally, a live attenuated vaccine could even be madecompatible with current or experimental new diagnostics (Cirillo et al.,1995). Although recent studies with subunit vaccines have beenpromising, so far these vaccines conferred shorter survival than BCGwhen tested in M. tuberculosis aerogenically challenged guinea pigs,underlying the problems to be solved with these types of vaccines(Baldwin et al., 1998). However, subunit vaccines also may be importantfor disease control in immunodeficient individuals or in areas where thestandard skin test has diagnostic significance. Furthermore, a subunitvaccine may be useful to boost individuals previously vaccinated withBCG or those at risk of disease reactivation (Baldwin et al., 1998).

[0034] The generation of a new live-attenuated vaccine poses manyresearch challenges. Genetic systems to manipulate M. bovis BCG and M.tuberculosis offer the greatest promise for the development of new andeffective vaccines against mycobacterial infections. Methods for allelicreplacement and transposon mutagenesis in slow-growing mycobacteria havebeen reviewed (Pelicic et al., 1998). Using conditionally replicatinggenetic elements, two groups have reported major breakthroughs in thegenetic manipulation of M. tuberculosis (Bardarov et al., 1997; Pelicicet al., 1997). Methods to generate unmarked deletion mutations in M.smegmatis, BCG, and M. tuberculosis have been described (Pavelka &Jacobs, 1999). This technology coupled with the knowledge of the M.tuberculosis genetic blueprint (Cole et al., 1998) should provide novelstrategies for the rational development of live attenuated vaccines. Themost promising approach for vaccine development would be to start withvirulent M. tuberculosis microorganisms and generate attenuated mutants.These mutants should carry at least two attenuating deletion mutationsand no antibiotic resistant markers, so as to avoid unwanted reversionsor transfers of drug-resistance. It is contemplated that vaccine strainsgenerated by the method of the present invention will include one ormore additional mutations. Other important issues to consider are a) thedegree of attenuation: mutants that are too attenuated may not induceprotective immunity as it was the case for S. typhimurium phoP mutants(Fields et al., 1989); and b) whether the effect of the vaccine straincould be potentiated by co-administration of cytokines or by endowingthe engineered strain with the capability to produce cytokines orphagosome membrane disrupters as lysteriolysin. In this case, temporalexpression of these genes may be critical and may have to be regulatedby promoters solely active inside phagocytic cells, as for example thepromoter of the mig gene, first identified in M. avium (Plum &Clark-Curtiss, 1994) with a corresponding homologue (fadD19) found inthe M. tuberculosis genome sequence (Cole et al., 1998).

[0035] Candidate genes for attenuating mutations may be those directlyinvolved in the pathogenic process or those encoding housekeepingenzymes whose suppression may alter the ability of M. tuberculosis tosurvive and/or replicate within phagocytes. The best documented examplesof mutations leading to attenuated phenotypes in vivo are theinactivations or deletions of the M. tuberculosis genes erp (Berthet etal., 1998), hma (Dubnau et al., 2000), katG (Li et al., 1998), leuD(Hondalus et al., 2000), mgtC (Buchmeier et al., 2000), and the recentlydescribed cyclopropane synthetase gene pcaA (umaA2) involved in cordformation (Glickman et al., 2000), and purC (Jackson et al., 1999). Inaddition, a M. tuberculosis deletion mutant unable to synthesizemycobactin was generated and shown to be impaired for growth in a humanmacrophage cell line (De Voss et al., 2000). Additional candidate genesand previous work has been summarized elsewhere (Cole et al., 1998).Other candidates are unidentified mycobacterial genes such as thoseinvolved in the inhibition of phagosome acidification (Sturgill-Koszyckiet al., 1994; Via et al., 1998). It may be also possible to identify andinactivate genes responsible for subtle changes in the structure oflipoarabinomannan since these changes influence receptor-mediated uptakeand ultimately intracellular fate and overall virulence (Kang &Schlesinger, 1998). Another approach that has been followed for theattenuation of M. tuberculosis is to engineer a recombinant strainoverexpressing a global negative regulator of virulence genes. In thiscontext, it was found that a recombinant M. tuberculosis carrying adominant constitutively active iron repressor was attenuated in mice(Manabe et al. 1999). The above literature also describes standardmethods for testing candidate vaccine strains in animal model systemssuch as mice or guinea pigs.

[0036] It was discovered that M. smegmatis alrA mutants display anincreased susceptibility to bactericidal action of phagocytic cells suchas macrophages (Chacon, O., 2002, incorporated herein by reference.)Based on this finding, pathogenic species of mycobacteria having aninactivated alrA gene could be constructed using methods known in theart. Such strains would be useful for the generation of attenuatedlive-attenuated vaccine candidates against tuberculosis and other humanor animal diseases caused by mycobacteria such as leprosy and Crohn'sdisease and Johne's disease. Further, based on the discovery that alrAmutants are impaired in the ability to synthesize the basic buildingblock of cell walls, peptidoglycan, vaccine strains which undergospontaneous lysis in vivo can be constructed using methods known in theart. Lysis would allow for improved antigen delivery and containment ofthe vaccine strain.

[0037] To determine the effect of the inactivation of the alrA gene onthe intracellular survival of M. smegmatis in phagocytic cells,monolayers of human macrophages were infected with wild type, mutant andrecombinant control strains. Prior to infection, M. smegmatis strainswere grown in medium supplemented with D-alanine which results inapproximately the same rate of growth for all strains. Growth inmonolayers was carried out in absence of D-alanine to approximate theconditions expected in the host. Monolayers were infected with M.smegmatis strains at a multiplicity of approximately 10 bacilli percell, invasion was allowed to proceed for 2 h and extracellular bacteriawere removed. At 24, 48 and 72 h post-infection, the number ofintracellular surviving bacteria was determined as described inMaterials and Methods. As expected, the wild type strain mc²155displayed moderate survival in monolayers of adherent human macrophages,with about 5-fold reduction in the number of intracellular bacteriaafter 72 h of incubation of the monolayers. In contrast, the alrA mutantstrain TAM23 was less able to survive reaching a 5-fold reduction inintracellular bacilli at 24 h and 500-fold by 72 h. Thus, theinactivation of the alrA gene has a significant effect on the ability ofM. smegmatis to survive within phagocytic cells.

[0038] To verify that the decreased survival of the mutant strain wasdue to the inactivation of the alrA gene, M. smegmatis TAM23 wascomplemented with a wild type copy which was delivered into thechromosome using an integrating vector. Strain TAM23(pTAMU3), carryingboth the inactivated and wild type copies of the alrA gene, displayedwild type survival in human macrophages. This result confirmed that theinactivation of the alrA gene is responsible for the reduced survival ofTAM23. In addition, the effects of introducing selection markers on thesurvival of M. smegmatis in phagocytic cells were tested usingappropriate recombinant control strains. The kanamycin-resistant markerused to inactivate the alrA gene was introduced into the chromosome ofthe wild type strain via an integrating vector. The resultingrecombinant strain mc²155(pYUB178) displayed the same survival patternas the wild type strain indicating that the introduction of this markerdoes not affect the intracellular survival of M. smegmatis. Since thevector used to complement the mutant strain carries ahygromycin-resistant marker, strain TAM23 was also transformed with thisvector alone to generate the recombinant alrA mutant strainTAM23(pYUB412). In this case, the survival of this recombinant strainwas slightly affected by the introduction of this marker. At 24 hpost-infection, both TAM23 and TAM23(pYUB412) displayed a 5-foldreduction in intracellular survival; however at 48 and 72 hpost-infection, TAM23(pYUB412) displayed an intermediate level ofsurvival when compared to the wild type and TAM23 mutant strains. (FIG.4). Thus, the hygromycin resistant marker has an influence onintracellular survival at longer times post-infection. Nonetheless, theeffect is minor and significantly different from the complete recoveryof the survival of TAM23 conferred, at every time-point post-infection,by the hygromycin-resistant integrating vector pTAMU3 carrying the wildtype gene.

[0039] Method of Use: Identification of New Antimycobacterial Agents

[0040] In addition to new effective vaccines against mycobacteria, thereis also an urgent need to develop new antimycobacterial agents. Thesenew agents should be effective against these microorganisms withoutbeing toxic to the mammalian host. Drug therapy is the major approach totreat human mycobacterioses. Several primary and secondaryanti-mycobacterial agents are currently used to treat humantuberculosis. The Food and Drug Administration has approved thefollowing drugs as primary agents: ethambutol, isoniazid, pyrazynamide,rifampin, and streptomycin. Examples of approved second-line drugs are:amikacin, ciprofloxacine, cycloserine, ethionamide, andpara-aminosalicylic acid. Recommended treatments use combinationtherapies with three agents for at least six months. However, theseregimens are becoming ineffective due to the emergence of drugresistance strains, including the appearance of clinical isolates withmultiple resistances to various primary and secondary drugs (Edlin etal., 1992; Kochi et al., 1993; Musser & Barry 1998). Thus, there is anurgent need to develop new and more effective anti-tuberculosis drugs.

[0041] Recent studies on mycobacterial drug resistance have focused onthe study of specific biochemical targets of the primary and secondarydrugs (Musser et al., 1999). For a summary of the principal targetsidentified to date, see Chacon, Ph.D. Thesis 2002, incorporated hereinin its entirety. In all these cases, the common theme is that thesetargets are enzymatic proteins involved in key metabolic processes forthe Mycobacterium. Identification and biochemical analysis of theseproteins is then applied to the rational design of new and moreeffective anti-mycobacterial agents (Zhang & Telenti, 2000).Furthermore, genes encoding for some of these proteins may also serve astargets for the generation of attenuated strains that could be useful indeveloping effective vaccines against mycobacteria. This might be thecase with genes playing an important role in metabolic pathways that arenecessary for the biosynthesis of mycobacterial structural components.One of these genes, involved in the biosynthesis of peptidoglycan, isthe alrA gene. The present invention is equally applicable to othermycobacterial genes involved in the synthesis of D-alanine.

[0042] The inhibition of AlrA by DCS in a concentration-dependent mannerindicates that this enzyme serves as one of the drug targets (Caceres etal, 1997). However, the dispensability of the alrA gene for growth invitro suggests that it is not the main target responsible for thebactericidal effect of DCS. Since overproduction of the enzyme leads toincreased resistance to DCS, it appears that binding of DCS to AlrAprotects another target within the cell. This hypothesis predicts thatAlrA null mutants would be hypersusceptible to DCS. Thus, thecorresponding MICs for wild-type and TAM23 alrA mutant strains weredetermined.

[0043] For MIC tests, M smegmatis cells grown with and without n-alaninewere inoculated onto complete Middlebrook MADC broth without D-alanine.The MIC of DCS for the wild-type strain was 75 μg/ml, independent of thepresence of D-alanine in the original inoculum (Table 2). As expected,TAM23 was about 30-fold more sensitive (MIC, 2.56 μg/ml) when theinoculum was grown without D-alanine. Since TAM23 cells carry a mutationin a gene responsible for D-alanine biosynthesis, it is possible thatgrowth in the presence of D-alanine partially restores wild-type MICs ofDCS. To test this hypothesis, TAM23 was grown with D-alanine, harvested,washed extensively to prevent D-alanine carryover, and inoculated intothe MIC test cultures. These conditions resulted in a fourfold increasein the MIC (MIC, 10.2), still about 7.5 times lower than the MIC for thewild-type strain. As a control, the MICs of the unrelated drugsamikacin, ethambutol, and rifabutin were also determined. The MICs forboth wild-type and mutant strains were the same, independently of thepresence or absence of D-alanine in the medium used to grow the inoculato determine the MICs. The effect of the addition of D-alanine directlyinto the MIC test cultures was also determined. D-Alanine would beexpected to effectively compete with DCS and decrease thesusceptibilities of both strains to DCS. As expected, under theseconditions, the MIC of DCS for both wild-type and TAM23 strainsincreased dramatically to 1,200 μg/ml independently of prior growthconditions of the inocula. No significant differences were observed forthe other unrelated drugs, demonstrating that the effect was specificfor DCS. Complementation of strain TAM23 with the integrating vectorpTAMU3, which carries the wild-type air gene, resulted in a strain forwhich the MICs were identical to those for the wild type. TABLE 2Minimal inhibitory concentrations of M. smegmatis wild type and TAM23alr mutant strains to selected antimycobacterial agents determined withinocula grown with or without D-alanine. Minimal InhibitoryConcentration^(a) (MIC, μg/ml) Wild Type mc² 155 TAM23 alr MutantWithout With Without With Drug D-alanine^(b) D-alanine^(c) D-alanine^(b)D-alanine^(c) Amikacin 1.56 N.D. 1.56 1.56 D-Cycloserine 75.0 75.0 2.5610.2  Ethambutol 3.13 N.D. 3.13 3.13 Rifabutin 2.00 N.D. 2.00 1.00

[0044] This increased susceptibility indicated by the MIC growthinhibition data was also confirmed by the analysis of the bactericidalaction of DCS in broth cultures grown in absence of D-alanine. Pilotexperiments were performed with each strain to determine the optimal DCSconcentration that resulted in a strong bactericidal effect. In thefinal experiment, cultures were grown to an early exponential phase(OD₆₀₀ of ˜0.4), split in two, and DCS was added to one of thesesubcultures at 50 times the MIC. In absence of DCS, both the wild-typestrain mc²155 and the TAM23 mutant cells grew to a OD₆₀₀ of >2.0 andreached saturation at a cell density approximately above 5.0×10. Thesedata also confirmed the independence of M smegmatis air mutants onD-alanine for growth and further demonstrate that air mutants can growin absence of D-alanine at approximately the same growth rate aswild-type cells. In contrast, in the presence of DCS both cellsunderwent rapid death by lysis as revealed by both the drastic decreaseobserved in optical density and viable counts. Furthermore, the kineticof killing was similar for both the wild-type and mutant strains.However, it must be emphasized that considering absolute drugconcentrations, the effect on the mutant strain is observed at a30-fold-lower concentration than for the wild-type strain. This patternsuggests that the bactericidal action of DCS is due to the inhibition ofa more fundamental target different from AlrA.

[0045] An alternative explanation for the increased susceptibility ofthe null mutants to DCS is that the inactivation of the alrA genechanges the permeability of the cell wall to DCS. This alteration maynot be detectable by morphological studies. To test this hypothesis, amethodology to perform DCS uptake assays was developed. The resultsobtained were similar for both wild-type and mutant strains. D-alanineuptake assays using a standardized procedure obtaining similar resultsfor both strains was also carried out. Thus, a permeability change wasruled out as responsible for the observed phenotype of the null mutants.

[0046] The uptakes of D-alanine and DCS in both wild-type and mutantstrains were not significantly different, indicating that changes incell wall permeability are not a likely explanation for the DCSincreased susceptible phenotype of the alrA mutant strain. Thus, theincreased susceptibility of TAM23 cells to DCS is consistent with theexistence of multiple targets for DCS. Our previous studies identifiedD-alanine racemase as one of these targets (Caceres et al, 1997). Thelack of the racemase protein in the alrA mutant strain may lead to ahypersusceptible phenotype since more DCS would be required to inhibitboth the racemase and an additional target(s) in the wild-type strain.In contrast, the bactericidal effect of DCS suggests the existence ofanother lethal target. In this context, D-alanine ligase is anattractive candidate since this enzyme activity is also inhibited by DCS(David, 1969; Chacon, Ph.D. thesis). More importantly, the constructionof a conditionally lethal mutant bank led to the isolation of athermosensitive mutant impaired in this gene function (Belanger et al.,2000). Furthermore, increased DCS susceptibility may reflect analteration of the peptidoglycan structure of TAM23 cells, as a directconsequence of the inactivation of the alr gene. In this context, TAM23cells were also more susceptible to DCS than were wild-type cells whengrown in medium with D-alanine, but mutant cells grown without D-alaninedisplayed increased susceptible to both growth inhibition and thebactericidal action of DCS.

[0047] Based on the finding that inactivation of the alrA gene of M.smegmatis yielded a strain with increased susceptibility to D-alanineanalogs, the generation of Alr null mutants as vaccine candidates wouldbe possible using methods known in the art. The present inventionaccordingly seeks to provide novel live-attenuated mycobacteria asvaccines. Such vaccines would be especially effective for treatment ofimmunosuppressed patients, or others displaying signs of disease, withdrugs such as D-alanine analogs.

[0048] Method of Use: Use of D-Alanine Racemase Mutants to Identify NewPathways of D-Alanine Biosynthesis

[0049] It was discovered that M. smegmatis D-alanine racemase mutantscan synthesize D-alanine by verifying the presence of D-alanine withinthe internal amino acid pool of TAM23. To determine the gene(s)responsible for the alternative pathway of D-alanine biosynthesis, thefollowing procedure is followed: 1) a transposon mutant bank of TAM23 isconstructed, 2) mutants are screened for the ability to grow in thepresence or absence of D-alanine in the growth medium, 3) mutants unableto grow in the absence of D-alanine are identified by replica, 4) theregion containing the transposon is cloned, and 5) the gene inactivatedby the transposon is identified by DNA sequencing. Alternatively, aproteomic analysis of TAM23 can be performed before and after a shiftfrom medium with D-alanine to media without D-alanine. Proteins inducedunder the latter conditions are candidates for enzymes involved in analternative pathway of D-alanine biosynthesis. Protein spots aresequenced, and the coding gene(s) are identified and then inactivated toconstruct strains carrying multiple mutations. These strains areevaluated for their dependency on D-alanine for growth.

[0050] Based on the finding that TAM23 mutant of M. smegmatis wasindependent of D-alanine for growth, the inactivation of the alrA geneprovides a means to study alternate metabolic pathways related topeptidoglycan synthesis in pathogenic mycobacteria. The discovery thatmycobacteria possess a novel pathway of peptidoglycan synthesis, makespossible the exploitation, using methods known in the art, of such inthe generation of other attenuated strains and new antimycobacterialdrugs. The present invention accordingly seeks to provide novel AlrAnull mutant mycobacterial strains for use in determining alternatemetabolic pathways of D-alanine and/or peptidoglycan biosynthesis in theidentification of drug targets for development of new bactericidalagents for the treatment of mycobacterial diseases.

[0051] Method of Use: Use of a DCS Hyper-Susceptible Mutant to Screenfor Cell Wall Inhibitors

[0052] An alrA mutant can be used to screen for inhibitors ofpeptidoglycan synthesis following methods known in the art (Kamogashiraand Takegata, 1988, incorporated herein by reference).

EXAMPLES

[0053] The present invention is further described in the followingexamples, which are offered by way of illustration and are not intendedto limit the invention in any manner. Standard techniques well known inthe art or the techniques described below are utilized in the practiceof the present invention.

Example 1 Generation of M. smegmatis alrA Mutants

[0054] Bacterial strains, plasmids, and culture conditions. Bacterialstrains and plasmids used in this study are listed in Table 1. E. colistrains were grown at 37C in Luria-Bertani broth or agar. M. smegmatisstrains were routinely grown at 37C in Middlebrook 7H9 base broth oragar (BBL Microbiology Systems, Cockeysville, Md.) supplemented with0.5% bovine serum albumin fraction V (EM Science, Gibbstown, N.J.), 0.01M dextrose (Sigma Chemical Co., St. Louis, Mo.), 0.015 M sodiumchloride, and 0.2% glycerol (MADC). Broth medium was also supplementedwith 0.05% Tween go (Sigma), and solid medium was made with 1.5% BactoAgar (Difco Laboratories, Detroit, Mich.). When required, O-alanine(Sigma) was used at 50 mM. Liquid cultures were incubated with shakingat 200 rpm in an Innova 4300 rotary incubator (New Brunswick ScientificCo. Inc., Edison, N.J.). As needed, the following antibiotics were usedat the specified concentrations: ampicillin (Sigma), 50 μg/ml for E.coli; kanamycin A monosulfate (Sigma), 25 μg/ml for E. coli and 20 μg/mlfor M. smegmatis; hygromycin B (Roche Molecular Biochemicals,Indianapolis, Ind.), 100 μg/ml for E. coli and M. smegmatis. For someexperiments, M. smegmatis was grown in a broth minimal medium based onthe formulation of Zygmunt, 1963, as modified by Caceres, 1999.Components and final concentrations were 5.0 mM ammonium chloride,6.8×10⁻⁷ mM calcium chloride, 8.4×1O-7 mM cobalt(II) chloride, 22 mMdibasic potassium phosphate, 2.5×1O-10 mM ferric chloride, 21 mMglycerol, 2.4 mM magnesium sulfate, 1.0×10-5 mM manganese chloride, 16mM monobasic potassium phosphate, 4.9×10-6 mM pyridoxal hydrochloride,0.4 mM Tween 80, and 8.6×10-6 mM zinc sulfate. Individual chemicals werepurchased from Sigma.

[0055] Bacterial transformation. E. coli was transformed as previouslydescribed (Ausubel et al., 1990). For the generation of M. smegmatisalrA mutants, 50 ml of an early exponential phase culture of M.smegmatis mc2155 was washed twice and concentrated 100-fold in cold 10%ultrapure glycerol (Invitrogen Life Technologies, Carlsbad, Calif.).Concentrated cells were electroporated with approximately 5.0 μg of theBamH1-KpnI fragment of pTAMU2 carrying the inactivated alrA gene.Electroporation was carried out at 2,500 V. 100 μF, and 246 Ω in anelectrocell manipulator (model 600; BTX Inc., San Diego, Calif.).Electroporated cells were allowed to recover at 37C in MADC broth andplated on MADC agar supplemented with 50 mM D-alanine (Sigma) andkanamycin (20 μg/ml; Sigma). For the genetic complementation experiment,50 ml of an early-exponential phase culture of M. smegmatis TAM23 alrAmutant was electroporated with 1.5 mg of pTAMU3 DNA at 2,500 V, 25 μF,and 1,000 Ω in a Gene Pulser electrocell manipulator (Bio-RadLaboratories, Richmond, Calif.), as previously described (Foley et al.,1995). Transformants were selected on MADC agar supplemented withhygromycin (100 μg/ml; Roche).

[0056] Nucleic acid manipulations. Mycobacterial DNA was isolated by thestandard method using cetyl trimethyl ammonium bromide (Ausubel, 1990).Plasmid DNA was isolated by an alkaline lysis method as previouslydescribed (Sambrook, 1989), using a large-scale isolation kit (Promega,Madison, Wis.) as recommended by the manufacturer. DNA fragments usedfor plasmid construction in E. coli and for recombination experiments inM. smegmatis were purified by gel electrophoresis and recovered byabsorption to glass particles (GeneClean Bin 101, Vista, Calif.) asrecommended by the manufacturer. Standard procedures were followed forrestriction digestions, ligations, and agarose gel electrophoreses(Sambrook, 1989).

[0057] Amplification of the alrA gene was carried out with 50 ng of M.smegmatis genomic DNA template using primers NAN-1 and NAN-2 for 27cycles in a thermal cycler (Perkin-Elmer Gene Amp PCR System 2400; RocheMolecular Systems, Branchhurg, N.J.) as previously described (Caceres,1997). For Southern blotting analysis, approximately 3.0 μg of M.smegmatis genomic DNA was digested with SmaI, and DNA fragments wereseparated on a 0.8% agarose gel, subjected to an alkaline denaturingprocedure, and transferred to Biotrans nylon membranes (ICN Biomedicals,Inc., Costa Mesa, Calif.). Membranes were hybridized with a probecorresponding to the 1.9-kb BamHI/PvuII fragment containing thewild-type M. smegmatis alr gene, which was radiolabeled with [α-32P]dCTPusing the Rediprime DNA labeling II system (Amersham Pharmacia Biotech,Inc., Piscataway, N.J.) as recommended by the manufacturer.Prehybridization and hybridization were performed at 56C. Washes weredone under high-stringency conditions at 65C as previously described(Sambrook, 1989).

[0058] Gram staining and acid-alcohol resistance testing. M. smegmatiscells were stained by the crystal violet method using a Gram stain kit(Sigma) as recommended by the manufacturer. Acid-alcohol resistance wasdetermined by the Zielsi-Neelsen acid-fast procedure using the TB StainKit ZN (Difco), as recommended by the manufacturer.

[0059] Electron microscopy. Strains were grown to an optical density at600 nm (OD655) of approximately 1.0 in MADC-Tween, with or withoutD-alanine. Cells were harvested, washed twice with phosphate-bufferedsaline (PBS)-0.05% Tween, and fixed for 1 h in 2.5% bufferedglutaraldehyde, washed twice, and postfixed with 2.0% osmium tetroxide.After repeated washings in PBS, samples were dehydrated in a gradedseries of ethanol solutions, washed twice in propylene oxide, andembedded with Araldite resin. Thin sections were examined with a Philips201 transmission electron microscope (Philips Electron Optics,Eindhovetn The Netherlands) at an accelerating voltage of 60 kV.

[0060] D-Alanine racemase assays. Approximately 200-ml cultures of M.smegmatis mc2155 or TAM23 were grown in MADC-Tween with or withoutD-alanine until they reached an OD₆₀₀ of ca. 1.0. Cultures were washedtwice and concentrated 50-fold in 50 mM Tris-HCl (pH 8.0). Cells weresonicated on a salt-ice-water bath with a Vibra-Cell model VC600sonicator (Sonic and Materials, Inc., Danbury, Conn.). Sonication wascarried out for 2 min at 80% power output and 50% duty cycle, and in thepresence of 30% (vol/vol) type A-5 alumina (Sigma). The resulting activecell extracts were centrifuged at 4C in a JA-17 rotor (BeckmanInstruments, Inc., Fullerton, Calif.) for 30 mm at 15,000 rpm, dialyzedagainst 50 mM Tris-HCl(pH 8.0), and sterilized by filtration through a0.22-μ-poresize filter (Advantec MFS Inc., Pleasanton, Calif.). Proteinconcentration was determined by the DC assay (Bio-Rad) as recommended bythe manufacturer. Alr activity in the active cell extracts was assayedin the direction of the conversion of L-alanine into D-alanine by amodification of the coupled-spectrophotometric method previouslydescribed (Caceres, 1999). To start the reactions, active crude cellextracts were added to 1.0 ml of prewarmed mixtures containing 50 mMTris-HCl (pH 8.0), 0.1 mM pyridoxal phosphate (Sigma), and 15 mML-alanine (Sigma). After 15 min of incubation at 37C, when theconversion of substrate into product remains linear, reactions werestopped by boiling for 10 min. Subsequently, 1 U of d-amino acid oxidase(Calzyme, San Lois Obispo, Calif.), 0.2 mM NADH (Roche Laboratories),and 10 U of rabbit muscle lactic dehydrogenase (Sigma) were added. Thecoupled reaction was measured by the change in absorbance at 340 nmafter 5 h of incubation at 37C. All samples were measured in triplicate.Specific activities (in micromoles of consumed substrate minute-1milligram-1) were calculated as previously described (Caceres et al,1997).

[0061] LDH assays. L-Lactate dehydrogenase (LDH) activity endogenous tocrude cell extracts of M. smegmatis was measured in the direction of theconversion of pyruvate into lactate coupled to the oxidation of NADH aspreviously described in the Worthington enzyme manual (WorthingtonBiochemical Corp., Lakewood, N.J.). Crude cell extracts were added to1.0 ml of prewarmed mixtures containing 50 mM Tris-HCl (pH 8.0), 1.0 mMsodium pyruvate (Sigma), and 0.2 mM NADH (Roche). The change inabsorbance at 340 nm was measured after 1 h of incubation at 37C. Allsamples were measured in triplicate. Specific activities (in micromolesof consumed substrate minute-1 milligram-1) were calculated as describedin the enzyme manual mentioned above by subtracting the backgroundchange in absorbance (obtained from boiled inactivated extractsprocessed in identical manner) from the change in absorbance obtainedwith the active cell extracts.

Example 2 Generation of M. tuberculosis alrA Mutants

[0062] The generation alrA mutants of M. tuberculosis were performedfollowing the procedures described in Example 1, with modifications asknow in the art (Braunstein et al., 2002; Bardarov et al., 1997; Pavelkaand Jacobs, 1999; Pelicic et al., 1997.)

Example 3 Drug Susceptibility Assays

[0063] MICs were determined by a microplate twofold dilution method, asdescribed by Takiff et al., 1996, with modifications. M. smegmatismc²155 and TAM23 cells were grown in 25 ml of MADC-Tween with andwithout 50 mM D-alanine to an OD₆₀₀ of approximately 1.0. Bacteria werewashed with PBS-Tween, and 105 CFU was inoculated onto each wellcontaining serial twofold dilutions of various antimicrobial agents.Plates were incubated at 37C, and visual inspection to determine MICswas carried out at 48 h. The MIC is defined as the lowest concentrationthat prevents observable bacterial growth and is determined by theconsistent result of three independent cultures, each assayed intriplicate.

[0064]M. smegmatis strains mc²155 and TAM23 were grown in MADC-Tweenbroth at 37C to an OD₆₀₀ of ca. 0.5. These starter cultures were used toinoculate 200 ml of MADC broth, and cells were then grown to an OD₆₀₀ ofca. 0.4. Two 50-ml aliquots of these exponentially growing cultures weretransferred to separate flasks. DCS was added to one of thecorresponding cultures, with the other serving as a growth control.Concentrations of DCS were 50 times the MIC for each strain (3.75 mg/mlfor mc²155 and 128 μg/ml for TAM23). All cultures were incubated at 37Cin a shaking incubator for 72 h. The OD₆₀₀ was measured for each cultureat 3, 6, 9, 12, 24, 48, and 72 h. Concurrently, aliquots were taken,serially diluted, and plated onto MADC agar in triplicate to determinenumbers of viable bacterial counts.

Example 4 D-Alanine Uptake Assays

[0065] For D-alanine uptake assays, the method described by David, 1971,was used. Cells exponentially growing in MADC-Tween (OD₆₀₀, ca. 1.0)were collected by centrifugation at 40C, washed once in ice-cold 50 mMTris-HCl buffer (pH 8.0) containing 0.05% Tween 80, and concentrated 20times in the same buffer. Samples were prewarmed for 10 min at 37C, andD-alanine was added to a final specific activity of 3.0 μCi of[14C]-1-D-alanine (ICN) per μmol asid 0.2 mM concentration. Samples wereplaced in a 37C water bath. Aliquots were taken at various times andimmediately placed on ice. The sample for each time point was divided inthree subsample triplicates of 0.5 ml and filtered through0.8-μm-pore-size membrane filters (Millipore Corp., Bedford, Mass.) in amanifold. Each filter was washed three times with 10 ml of cold buffer,dried under a heat lamp, and weighed. The cell-associated radioactivitywas determined in a liquid scintillation counter (Wallac 1410;Pharmacia, Piscataway, N.J.) using EcoLite scintillation cocktail (ICN).Uptake data were expressed as micromoles of D-alanine per milligram (drycell weight).

[0066] Since radiolabeled DCS is not commercially available, an uptakeassay was developed based on the colorimetric determination of thiscompound (Jones, 1956). Cells exponentially growing in MADC-Tween (OD₆₀₀of 1.0) were harvested at 4C by centrifugation, washed twice with water,and concentrated 40 times. Samples were prewarmed at 37C for 10 min, andDCS was added to a final concentration of 250 μg/ml (˜2.5 mM). Cellswere placed in a 37C water bath. Aliquots were taken at various timesand immediately placed on ice. After 20 min of incubation, clumping ofTAM23 cells was evident and uptake determinations became unreliable.Each time a point sample was washed twice with water, resuspended, andsonicated. Protein concentration was determined using the Bio-Rad DCassay, followed by protein removal through serial passages of both YM-10and YM-3 Centricon concentrators (Millipore Corp.). The concentration ofDCS was determined by measuring the OD620 after adding a specificcolor-developing reagent as previously described (Jones, 1956). Astandard curve was generated by diluting a DCS standard in a cellextract that was prepared from cells not exposed to DCS. Uptake datawere expressed as micromoles of DCS per milligram of protein. SinceD-alanine and DCS uptake assays do not measure the same parameters,reported values for each assay cannot be directly compared.

Example 5 Increased Susceptibility to Phagocytic Cells

[0067] Intracellular killing assays were carried out as describedpreviously with the following modifications (Bermudez and Young, 1988).Approximately 5×10⁵ monocytes were seeded on each well of a 24-welltissue culture plate (Sigma). Plates were incubated for 2 h at 37 C in5% CO₂. Supernatant fluids with nonadherent cells were removed and thewells were washed twice with 1.0 ml of prewarmed HBSS. The adherentmonocytes were maintained in culture for 7 days with 1.5 ml of RPMI 1640(Gibco) supplemented with 2 mM L-glutamine (Sigma) and 10%heat-inactivated fetal bovine serum (Sigma). Culture media was replacedevery 48 hours. Each well was inoculated with approximately 5.0×10⁶ CFUof bacteria. After 2 h, macrophage monolayers were washed three timeswith HBSS to remove extracellular microorganisms. Some wells were lysedimmediately with 0.5 ml of sterile water for 10 min and 0.025% SDS(Sigma) in HBSS for additional 10 min. SDS was subsequently inactivatedwith 0.1% bovine albumin (Sigma). The lysate was serially diluted andplated onto Middlebrook 7H9 based agar (BBL) supplemented with 0.5%fraction V albumin (EM Science), 0.01 M dextrose (Sigma), 0.015 M sodiumchloride (Sigma), 0.15% glycerol (Sigma) and 1.5% Bacto agar (Difco).The same procedure was done on two other sets of wells at 24, 48 and 72hours. Three wells were carried out per experimental group, in each ofthree independent experiments. It is to be noted that a preliminaryexperiment using 20 μg/ml of amikacin during the invasion step to killextracellular bacteria yielded similar results, demonstrating theeffectiveness of the washing procedure.

[0068] While the invention has been disclosed in this patent applicationby reference to the details of preferred embodiments of the invention,it is to be understood that the disclosure is intended in anillustrative rather than in a limiting sense, as it is contemplated thatmodifications will readily occur to those skilled in the art, within thespirit of the invention and the scope of the appended claims.

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What is claimed and desired to be secured by Letters Patent is asfollows:
 1. A vaccine composition comprising a live-attenuatedmycobacterium, wherein said live-attenuated mycobacterium comprises aD-alanine racemase mutant of a pathogenic species which displaysincreased susceptibility to an antimycobacterial agent.
 2. The vaccineof claim 1, wherein said pathogenic species of mycobacterium is selectedfrom the group consisting of Mycobacterium tuberculosis, Mycobacteriumavium, Mycobacterium bovis, Mycobacterium africanum, Mycobacteriumparatuberculosis, and subspecies thereof.
 3. The vaccine of claim 1,wherein said antimycobacterial agent is selected from the groupconsisting of D-cycloserine and other D-alanine analogs.
 4. A vaccinecomposition comprising a live-attenuated mycobacterium, wherein saidlive-attenuated mycobacterium comprises a D-alanine racemase mutant of apathogenic species which displays increased susceptibility tobactericidal action of phagocytic cells.
 5. The vaccine of claim 4,wherein said pathogenic species of mycobacterium is selected from thegroup consisting of Mycobacterium tuberculosis, Mycobacterium avium,Mycobacterium bovis, Mycobacterium africanum, Mycobacteriumparatuberculosis, and subspecies thereof.
 6. The vaccine composition ofclaim 1, wherein said live-attenuated pathogenic species ofmycobacterium further comprises one or more additional mutations whereinsaid live-attenuated mycobacterium is rendered dependent on D-alaninefor growth.
 7. The vaccine of claim 6, wherein said pathogenic speciesof mycobacterium is selected from the group consisting of Mycobacteriumtuberculosis, Mycobacterium avium, Mycobacterium bovis, Mycobacteriumafricanum, Mycobacterium paratuberculosis, and subspecies thereof. 8.The vaccine of claim 6, wherein said antimycobacterial agent is selectedfrom the group consisting of D-cycloserine and other D-alanine analogs.9. A vaccine composition comprising a live-attenuated mycobacterium,wherein said live-attenuated mycobacterium comprises a D-alaninedependent mutant of a pathogenic species said species further comprisingincreased susceptibility to bactericidal action of phagocytic cells. 10.The vaccine of claim 9, wherein said pathogenic species of mycobacteriumis selected from the group consisting of Mycobacterium tuberculosis,Mycobacterium avium, Mycobacterium bovis, Mycobacterium africanum,Mycobacterium paratuberculosis, and subspecies thereof.
 11. The vaccineof claim 6, comprising a single D-alanine ligase gene under the controlof an inducible promoter, said promoter controlling the expression ofD-alanyl-D-alanine of a pathogenic species, wherein said pathogenicspecies displays increased susceptibility to an antimycobacterial agent.12. The vaccine of claim 11, wherein said pathogenic species ofmycobacterium is selected from the group consisting of Mycobacteriumtuberculosis, Mycobacterium avium, Mycobacterium bovis, Mycobacteriumafricanum, Mycobacterium paratuberculosis, and subspecies thereof. 13.The vaccine of claim 11, wherein said antimycobacterial agent isselected from the group consisting of D-cycloserine and other D-alanineanalogs.
 14. A method for screening antimycobacterial agents that aresynergistic with peptidoglycan inhibitors comprising use of AlrA nullmutant mycobacteria.
 15. A method for identifying novel genes in thepathways of D-alanine biosynthesis, comprising: constructing atransposon mutant bank of TAM23, screening mutants for the ability togrow with the presence or absence of D-alanine in the growth medium,identifying mutants unable to grow in the absence of D-alanine, cloningthe region containing the transposon, and identifying the geneinactivated by the transposon by DNA sequencing.
 16. A pathogenicmycobacterium comprising an inactivated alrA gene, wherein saidmycobacterium is independent of D-alanine for growth.
 17. The pathogenicmycobacterium of claim 16, wherein said pathogenic mycobacterium isselected from the group consisting of Mycobacterium tuberculosis,Mycobacterium avium, Mycobacterium bovis, Mycobacterium africanum,Mycobacterium paratuberculosis, and subspecies thereof.